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Bax is upregulated by p53 signal pathway in the SPE B-induced apoptosis

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Abstract

We identify integrin αvβ3 and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S (SPE B mutant, glycine at residue 308 is changed to serine), which interacts with Fas only, in our previous study. Here, we explore the signal pathways that regulate proapoptotic protein expression after SPE B stimulation. We find that both SPE B and G308S can stimulate the serine phosphorylation of p53, and p53 phosphorylation is inhibited by the anti-Fas antibody but not by anti-αVβ3 antibody. p38 inhibitor and siRNA decrease the activation and translocation of p53 into the nucleus, which executes its transcription activity. These results indicate that after SPE B treatment, p53 is activated and p38 is the upstream of p53. p38 siRNA also decreases the binding of p53 to the bax promoter and interferes with the association of p53 and STAT1. p53, p38, and STAT1 siRNAs downregulate SPE B-induced Bax expression. This shows that SPE B activates the bax promoter via p38/p53 signal pathways through the Fas receptor, and that STAT1 acts as a coactivator of p53. In addition, p38 and p53 siRNAs inhibit SPE B-induced apoptosis. This is consistent with the findings that SPE B upregulates Bax expression through p38/p53 signal pathways that enhance cell apoptosis.

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Acknowledgment

We thank Jonathan Courtenay for editorial assistance.

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Authors and Affiliations

  1. Department of Otolaryngology, National Cheng Kung University Hospital, Tainan, Taiwan

    Wei-Ting Lee

  2. Departments of Biochemistry, Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan, 701, Taiwan

    Chia-Wen Chang

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Electronic supplementary material

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Supplement 1

. The level of Bax is not upregulated in p53 null H1299 cell. H1299 cell lysates are extracted and probed with an anti-p53 antibody. The membrane is then reprobed with an anti-β-actin antibody (a). After transfection with p53 reporter, cells (2 × 105) are treated with or without SPE B (20 μg/ml) and the luciferase activity is measured. Data are the means ± SEM of triplicate cultures (b). Cells (3 × 106) are treated with or without SPE B (20 μg/ml) for 5 h. Total RNA is prepared from cells and subjected to RT-PCR analysis of Bax mRNA expression. The cDNA is amplified with specific oligos for Bax and β-actin (c). Cells (2 × 105) are treated with or without SPE B (20 μg/ml) for 10 h. Total cell lysates are probed with an anti-Bax antibody. The membrane is then reprobed with an anti-β-actin antibody (d)

Supplement 2

. Bax translocation into mitochondria is not regulated by p53, and no p53 translocation into mitochondria is detected after SPE B treatment. Cells (2 × 105) are incubated with SPE B (20 μg/ml) with or without siRNA for various times. The mitochondria and cytosol extracts are probed with an anti-Bax antibody (a) or anti-p53 antibody (b). The membrane is then reprobed with an anti-β-actin antibody

Supplement 3

. The mRNA and protein levels of p53 are not increased after SPE B stimulation. Cells (3 × 106) are treated with SPE B (20 μg/ml) for various times. Total RNA is prepared from A549 cells and subjected to RT-PCR analysis of mRNA expression. The cDNA is amplified with specific oligos for p53 and β-actin (a). Cells (2 × 105) are treated with SPE B (20 μg/ml) for various times. Total cell lysates are probed with an anti-p53 antibody. The membrane is then reprobed with an anti-β-actin antibody (b)

Supplement 4

. The mRNA and protein levels of PUMA are not upregulated in the presence of SPE B. Cells (3 × 106) are treated with SPE B (20 μg/ml) for various times. Total RNA is prepared from A549 cells and subjected to RT-PCR analysis of mRNA expression. The cDNA is amplified with specific oligos for PUMA and β-actin (a). Cells (2 × 105) are treated with SPE B (20 μg/ml) for various times. Total cell lysates are probed with an anti-PUMA antibody (b). The membrane is then reprobed with an anti-β-actin antibody

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Lee, WT., Chang, CW. Bax is upregulated by p53 signal pathway in the SPE B-induced apoptosis. Mol Cell Biochem 343, 271–279 (2010). https://doi.org/10.1007/s11010-010-0522-6

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